5th Annual PRiME Symposium Abstracts
New Frontiers in Precision Medicine: Unleashing the Potential
Abstract #1
Employing first in class covalent class I selective HDAC inhibitors in targeting Niemann-Pick type C
Diaa Abdallah, PRiME Fellow 2024, University of Toronto Mississauga
Although rare, Niemann Pick Type C (NPC) is a progressive, genetic lysosomal storage disorder where patients have a life expectancy of <20 years due to the lack of rationally designed therapies. Recent evidence has emerged demonstrating the importance of histone deacetylase (HDAC) inhibition in impeding the cholesterol storage defect in NPC, thereby playing a key role in NPC progression. HDACs, particularly Class I HDACs (HDACs 1, 2, 3), are epigenetic modulators that have shown in vivo promise against NPC cholesterol accumulation when inhibited. Currently, there are 7 Class I HDAC inhibitors (HDACi) in clinical trials. However, all Class I HDACi in the clinic are pan-inhibitors that are plagued with off-target effects, leading to toxicities. HDAC3 has been specifically shown to be significantly dysregulated in NPC, making it an attractive therapeutic target. Here, we are exploring the utility of introducing covalent warheads to generate more efficacious compounds. This program is geared toward synthesizing, and evaluating the physico-chemical properties of HDAC3 covalent inhibitors in NPC, particularly in an in-vivo model.
Abstract #2
Lipid Nanoparticles as a Safe Vehicle for Fetal Gene Therapies: A Machine Learning Model for Placental Transport
Amr Abostait, Unity Health Toronto
Congenital disorders are a considerable challenge in maternal-fetal health. While gene therapies could restore normal development and offer definitive cures, there’s a need for safe and efficient gene delivery vehicles to fetal organs during pregnancy. Although lipid nanoparticles (LNPs) have been regulatory approved for vaccines and liver delivery, their interaction with the placental barrier and fetal organs is still understudied.
In this study, using microfluidic synthesis, we optimized a library of LNPs to achieve high placental transport. Utilizing the maternal-fetal transfer of passive immunity via IgG transport, the rate of LNPs transport was dependent on the IgG orientation on the surface of LNPs. Components of the LNPs, such as the PEGylated lipid type, PEG end-functional group, and the ionizable lipid, had the highest effect on transport. Syncytialization or trophoblast fusion significantly increased the transport rates for different formulations, suggesting a change of nanoparticles placental transport at the varying stages of pregnancy. Using hundreds of LNPs transport data points, from fifty-two formulations, a machine learning (ML) model was developed using the XGBoost algorithm, and it has identified the top features driving LNPs placental transport.
Moreover, Small interfering RNA (siRNA) transfection to fetal lung fibroblast was optimized by manipulating the cationic lipid percentage and the lipid to siRNA ratio. Finally, LNPs toxicity was assessed in a tracheal occlusion lung explant model. Fetal lungs showed normal airway development in histological staining and no effect on the Apoptosis and Proliferation markers after exposure to different LNPs.
Utilizing machine learning has enabled a mechanistic understanding of LNPs placental transport and delineated the top transport features for consideration. This research represents a noteworthy step towards establishing the safety and efficacy of LNPs delivery to fetal organs, exploring a new route for treating deadly congenital diseases.
Abstract #3
Developing a molecular staging stratification system for T-cell lymphomas
Mary Agopian, University of Toronto Faculty of Medicine
Bone marrow aspiration and biopsy (BMAB) is the gold standard for diagnosing T-cell lymphomas. However, T-cell lymphomas report lower bone marrow infiltration rates, leading to delayed diagnosis, treatment delays, and added patient burden. T-cell receptor repertoire sequencing (TCR-seq) presents a promising alternative for diagnosing T-cell lymphoma. However, it faces quantitative challenges affecting its ability to monitor and quantify T-cell populations. These challenges include PCR bias, sequencing depth variation, and read count fluctuations. To address these diagnostic challenges, we propose the incorporation of tailored spike-ins to TCR-seq. These spike-ins would normalize PCR biases and fluctuations, enabling the establishment of a robust and quantitative molecular staging system for monitoring and prognosis.
To create the tailored spike-ins, we started by modeling the biophysical properties characteristic of TCR loci sequencing. We then sampled from this framework using Latin hypercube sampling. Finally, the spike-in sequences were generated based on this established framework using a homogeneous zero-order Markov.
Our results showed that different TCR chains exhibited varying biophysical properties. As a result, we would have to develop distinct spike-in controls for each chain. However, our spike-ins are specifically tailored for the TCR-ß chain, the most frequently analyzed TCR chain in clinical settings. Additionally, the TCR region encompasses various recombinatorial regions with distinct biophysical properties. Therefore, separate characterization of each region was performed and assembled into an encompassing schematic. Finally, Latin hypercube sampling from this framework showed that stochastic sampling effectively captured the distinct biophysical properties exhibited by the TCR loci, facilitating the construction of robust and tailored spike-in controls.
In conclusion, our study introduces a novel approach to quantifying TCR-seq through incorporating tailored spike-ins to overcome quantitative challenges in T-cell lymphoma diagnosis via TCR-seq. These spike-ins, particularly designed for the TCR-ß chain, offer a promising solution for enhancing the accuracy and reliability of T-cell population monitoring and prognosis in clinical settings.
Abstract #4
Novel affinity-based electrochemical biosensor using stainless steel for early diagnosis of ovarian cancer
Soha Ahmadi, University of Toronto Faculty of Arts and Science
Electrochemical techniques are ideal for developing point-of-care medical devices due to their high sensitivity, fast response, and miniaturization potential. However, a major challenge in developing these devices is the issue of contact of artificial materials with biological fluids such as blood, serum, and urine. In this context, two key factors are involved: first, the deleterious effects that may be induced by artificial surfaces due to bio-incompatibility leading to unwanted reactions; and second, the fouling of nontarget molecules on the recognition surface of the biosensor, a phenomenon known as non-specific adsorption (NSA). The significant level of NSA associated with the gold electrode limits its application for fabricating diagnostic biosensors. Any biomolecules with a thiol group, such as cysteine-containing proteins in the biofluid, can bind to the gold electrode leading to false results. To overcome NSA, we used a non-conventional material for the fabrication of the working electrode. We have developed an affinity-based electrochemical biosensor using medical-grade stainless steel electrodes following a unique and novel strategy using silane-based interfacial chemistry to detect lysophosphatidic acid (LPA). LPA is a highly promising biomarker, which was found to be elevated in 90% of early stages of ovarian cancer and gradually increases as the disease progresses to later stages. Stainless steel is cost-effective compared to gold; therefore, the working electrode can be fabricated with a larger surface area to enhance the sensitivity of the biosensor while keeping costs low. The biorecognition surface was developed using the affinity-based gelsolin-actin system, which was previously investigated by our group to detect LPA using fluorescence spectroscopy. We have demonstrated the ability of this label-free biosensor to detect LPA in goat serum with a detection limit of 0.7 µM. This proof-of-concept study shows the potential of using our biosensor for the early diagnosis of ovarian cancer.
Abstract #5
Systematically and proactively testing variant effects for the autoimmune regulator gene
Anna Axakova, University of Toronto Faculty of Medicine
Pathogenic variants in the Autoimmune Regulator (AIRE) gene cause Autoimmune Polyendocrine Syndrome Type 1 (APS-1), a rare primary immunodeficiency disease with symptoms including chronic mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease. AIRE increases the expression of tissue-specific genes in the niche of developing T cells, thus triggering the elimination of emerging self-reactive T-cells and preventing autoimmunity. APS-1 diagnosis by AIRE sequencing is increasingly common, and earlier diagnosis can benefit patients. However, two thirds of clinical variants reported are missense, and more than half of reported clinical missense variants found to be “of unknown significance”. Testing variant function in cell-based assays can provide strong evidence for variant classification, but these tests are carried out reactively, often months or years after a variant’s first observation within a patient. By contrast, proactively assessing the function of all possible missense variants could provide immediate evidence for genetic diagnosis, even for never-before-seen variants. Here we describe the first saturated sequence-function map of AIRE missense variants, using an insulin promoter-driven reporter assay. Our sequence-function map of AIRE, which agrees with biochemical expectations and current pathogenicity annotations, provides proactive evidence for 219 variants of unknown significance, with potential to improve patient outcomes by providing more rapid and definitive genetic diagnoses of APS-1.
Abstract #6
Developing an organ-on-a-chip model of the human knee for drug testing purposes
Lauren Banh, University of Toronto Faculty of Engineering
Osteoarthritis (OA) is a degenerative joint disease affecting one in seven Canadians with no effective treatments that are readily available. This is partly because multifactorial parameters are not adequately represented in conventional laboratory models. Thus, we are using novel organ-on-a-chip engineering techniques to develop “cartilage-on-a-chip” (CoC) and “synovium-on-a-chip” (SoC) models for capturing OA hallmarks.
For the CoC model, we fabricated a device that compresses OA patient-derived articular cartilage biopsies through in-house micromilling fabrication techniques. The tissue was subjected to several hours of hyperphysiological (30%) depth compression at one hertz and accompanied by a rest period over fourteen days. The degree of cartilage degradation was measured by sulphated glycosaminoglycans (sGAG) in the conditioned medium and Safranin-O staining for proteoglycan content. For the SoC model, human synovial microvascular endothelial cells (HSyMVEC) with OA fibroblast-like synoviocytes (FLS) were co-cultured in a three-dimensional fibrin hydrogel for seven days. Changes in cell organization and lumen formation were monitored through confocal microscopy.
In the CoC model, mechanical compression increased the released sGAG concentration and decreased proteoglycan content relative to static unloaded controls, demonstrating that OA-like traits were induced by hyperphysiological compression. Biological readouts such as cartilage gene expression and degraded extracellular matrix protein concentrations are currently being added to the panel. In the SoC model, HSyMVEC spontaneously self-assembled into vascular-like networks with the support of periphery FLS by day three of culture and collapsed by day seven. Ongoing experiments are being done to optimize vascular network stability by adjusting FLS and HSyMVEC cell concentration ratios before integrating flowing immune cells through the vasculature.
Overall, CoC and SoC are more physiologically relevant joint models of mechanical cartilage degradation and synovial inflammation, respectively. These modules will be valuable tools for studying OA pathophysiology, and potential drug screening purposes when combined to reflect a “joint-on-a-chip” model.
Abstract #7
Defining the allosteric activation path for ClpP
Marim Barghash, University of Toronto Faculty of Medicine
Marim M. Barghash1,#, Mark F. Mabanglo1#, Dmytro Brozdnychenko3#, Samuel E. Hoff 4, Siavash Vahidi 3*, Massimiliano Bonomi 4*, and Walid A. Houry1,2*
1Department of Biochemistry, University of Toronto, Toronto, Canada
2Department of Chemistry, University of Toronto, Toronto, Canada
3Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada
4 Department of Structural Biology and Chemistry, Institut Pasteur, Paris, France
#Co-first authors, *co-corresponding authors
Molecular chaperones and proteases exist in all organisms where they play a critical role in maintaining cellular protein homeostasis. ClpP is one such protease present in both bacteria and eukaryotes. It is composed of fourteen identical subunits that typically assemble as stacked heptameric rings to form a hollow barrel-like structure with 7-fold symmetry. Chemical interference may be used to activate ClpP and dysregulate its function, resulting in the unregulated proteolysis of non-substrate proteins, causing cell death. As such, targeting ClpP has recently emerged as a promising avenue for the development of novel antimicrobial drugs. Classical activators bind in the hydrophobic sites of ClpP, while more recently, other activators have been seen to bind in the active sites. Here, we identified synthetic compounds that are able to bind in both sites by utilizing protease degradation assays and X-ray crystallography. We also solved the first structure of a fungal ClpP, both bound and unbound to Dioctatin, a small molecule activator produced in Streptomyces. Dioctatin binds both hydrophobic and active sites of ClpP. Inspired by this phenomenon, we defined the allosteric pathway for ClpP activation by using hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Taken together, this work advances our understanding of ClpP allostery, which can aid in drug design and development efforts in the future.
Abstract #8
An Acid-Responsive Polymer Additive Increases RNA Delivery Efficiency from Lipid Nanoparticles
Timothy Cheung, University of Toronto Faculty of Arts and Science
Lipid nanoparticles (LNP) are the most clinically advanced RNA delivery technology, but their efficiency is hampered by limited release of RNA from the nanoparticle after endosome disruption. As nanoparticle dissociation and subsequent RNA release after endocytosis have previously been demonstrated to increase biological effect, we hypothesized that the addition of an acid-responsive RNA releasing polymer to LNPs would facilitate these processes and improve RNA delivery. To test our hypothesis, we synthesized the polymer, then characterized the polymer’s ability to complex with RNA at varying pH. We observed that at pH 7.4, the polymer was cationic and complexed with RNA, but at endosomal pH, the polymer was irreversibly converted to neutral and had lowered affinity to RNA. The polymer was then formulated as an additive into clinically relevant LNP formulations by microfluidic mixing. The produced polymer-lipid hybrid nanoparticles (PLNPs) had high RNA encapsulation and diameters of approximately 150 nm. RNA delivery from PLNPs was then tested by siRNA-mediated knockdown of the reporter gene firefly luciferase (FLuc) in an ovarian cancer cell line. When added to the clinically used Onpattro LNP formulation, the polymer lowered the IC50 of anti-FLuc siRNA 5.4-fold. Similarly, the polymer lowered the IC50 3-fold when added to the Moderna LNP formulation and 5-fold for the Pfizer formulation. When the acid-responsive polymer was replaced with a chemically similar, but acid-inert polymer, the PLNPs no longer had an enhanced effect over the conventional formulations, demonstrating that acid-responsive RNA release was important to the delivery mechanism. The PLNP formulation was then used to knockdown the drug resistance associated gene cyclin E1 in a breast cancer cell line, demonstrating that the strategy was applicable to multiple cell lines and various genes. Thus, our novel polymer represents a versatile strategy to improve RNA delivery efficiency from multiple LNP formulations.
Abstract #9
Immunomodulatory Potential of Extracellular Vesicles Derived from Enhanced Mesenchymal Stromal Cells
Griffin Copp, University of Toronto Faculty of Engineering
Extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) have emerged as primary therapeutic agents due to their cargo including nucleic acids, proteins, lipids, and signalling molecules. Despite the mixed clinical success of MSC therapies, recent recognition of EVs as primary therapeutic agents warrants investigation into strategies enhancing their efficacy. This study explores the impact of various non-genetic cell-enhancing techniques on MSC-secreted EVs, explicitly focusing on their ability to polarize macrophages toward an anti-inflammatory (M2) phenotype. Understanding these immunomodulatory properties is crucial for optimizing cell-based therapeutic strategies.
Adipose tissue-derived MSCs are enhanced through inflammatory priming, transient hypoxic conditioning, or transient 3D culturing. Small EVs are isolated using differential ultracentrifugation and characterized through nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Bradford assay (BCA), and Western blot. A macrophage polarization assay, assessed via real-time qPCR, is employed to determine the immunomodulatory capacity of EVs. Proteomic differences between enhanced EVs and controls can then be further elucidated through liquid chromatography with tandem mass spectrometry.
Preliminary data reveals a distinct clustering of enhanced MSC-EVs in a principal component analysis of macrophage polarization read-outs, indicating altered immunomodulatory effects compared to naive MSC-EVs. TEM images verify the morphology of small EVs, while NTA demonstrates particle quantity and size distribution (peaking < 200nm). BCA quantifies protein content, and Western blot validates the presence of exosome-specific markers (CD-9, -63, -81).
This study sheds light on the increased immunomodulatory potential of MSC-derived EVs following various cell-enhancing techniques. The observed differences in macrophage polarization suggest a nuanced interplay between MSC modifications and EV functionality. These findings contribute valuable insights for developing consistently therapeutic cell products, emphasizing the pivotal role of EVs in cellular strategies for clinical applications.
Abstract #10
Precision targets and therapies for eye disease
Tim Corson, University of Toronto Leslie Dan Faculty of Pharmacy
Abnormal blood vessel growth (neovascularization) is a defining feature of eye diseases like proliferative diabetic retinopathy, wet age-related macular degeneration, and retinopathy of prematurity, as well as ocular cancers. Together, these are a top cause of vision loss and blindness worldwide. Existing drugs all target the same mechanism: vascular endothelial growth factor (VEGF) signaling. Not all patients respond to these drugs, and they must be injected into the eye. Thus, there is a need for next-generation, precision therapies for these diseases delivered topically or orally. Our laboratory, recently relocated to the University of Toronto, explores drug discovery for eye disease using forward and reverse chemical genetic approaches. Using forward chemical genetics, we identified targets of antiangiogenic small molecules. We found the heme synthesis enzyme ferrochelatase (FECH) as a target of an antiangiogenic natural product called cremastranone. We are characterizing the unexpected roles of heme synthesis in mediating angiogenesis via cellular energetics. We have also identified the first drug-like FECH inhibitor by high-throughput screening and medicinal chemistry optimization, and repurposed an old drug called griseofulvin for ocular use. Using cell-based angiogenesis assay screening, we identified another novel antiangiogenic compound called SH-11037. We identified its target as the bioactive lipid modulating enzyme soluble epoxide hydrolase (sEH), and are exploring the role of sEH and its epoxylipid substrates in ocular angiogenesis in vitro and in vivo. We are also using reverse chemical genetics to find new and improved inhibitors of the redox-modulating transcriptional regulator, Ref-1, while exploring how this protein is important in eye disease. A first-generation Ref-1 inhibitor is entering a Phase III trial for diabetic retinopathy. Our lab collaborates with synthetic and natural products chemists, cancer biologists, formulations experts, and clinicians to move our discoveries towards commercialization and the clinic.
Abstract #11
NM001 prevents septic-induced myocardial dysfunction by targeting the inflammatory cytokines TNFa and IL-6
Amin Ektesabi, Unity Health Toronto
Background: Patients with severe, sepsis can develop acute heart failure characterized by left ventricular dilatation and decreased contractility. Inflammatory mediators, including TNF-alpha, IL-6 and Il-1beta contribute to ventricular remodeling and activating pro-inflammatory pathways in sepsis. However, therapeutic interventions for polymicrobial sepsis are lacking. We hypothesize that administering our RNA based drug, NM001, would reduce inflammatory cytokine production and improved cardiac function and mortality rate overall.
Methods: Polymicrobial sepsis was induced in C57b6 mice via cecal ligation and perforation (CLP) surgery, while mice undergoing sham surgery served as the control group. NM001, RNA negative control, or saline were administered IV 6 hours post-CLP. At 48 and 168 hrs post-CLP, following echocardiographic and hemodynamic assessments, the mice were sacrificed, and the hearts collected for biochemical, and molecular analysis.
Results: Administration of NM001 to septic mice 6 hrs post-CLP increased survival, improved echo-derived indices of cardiac function (left ventricular ejection fraction, fractional shortening), increased bacterial clearance and reduced inflammation when compared to septic mice receiving a scrambled control miR. In addition to TNF-alpha, bioinformatic analysis, confirmed by luciferase assays, identified IL-6 as a specific target of NM001 in cardiac tissue. In septic patients, the expression of the target RNA and their target inflammatory cytokines were decreased in human circulatory blood and in autopsied hearts. Which NM001 tries to replace.
Conclusion: The results demonstrate that delivery of NM001 improves outcomes in sepsis-induced myocardial dysfunction. Thus, NM001 serves as a potential therapeutic target for prevention and prognostic reflection in sepsis-induced myocardial dysfunction.
Abstract #12
Identification and characterization of molecules with novel antifungal activity against Candida albicans.
Sara Fallah, University of Toronto Faculty of Medicine
Sara Fallah1, William J. Steinbach2,3, Joseph Heitman4, John A. Porco Jr.5, , Lauren Brown4, Partha P. Nag6, Luke Whitesell1, Nicole Robbins1, and Leah E. Cowen1
Affiliations
1 Department of Molecular Genetics, University of Toronto, Toronto, Canada
2 Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, USA
3 Arkansas Children's Research Institute, Little Rock, USA
4 Department of Molecular Genetics and Microbiology, Duke University, Durham, USA
5 Department of Chemistry and Center for Molecular Discovery (BU-CMD), Boston University, USA
6 Chemical Biology and Therapeutics Science (CBTS), The Broad Institute of MIT and Harvard, USA
Infection with Candida albicans, one of the most prevalent fungal pathogens of humans, causes a diverse range of diseases extending from superficial infections to deadly systemic mycoses. Currently, only three major classes of antifungal drugs are available to treat systemic infections: azoles, polyenes, and echinocandins. Alarmingly, the efficacy of these antifungals against C. albicans is hindered both by basal tolerance towards the drug and the development of resistance mechanisms such as alterations of the drug’s target, modulation of stress responses, and overexpression of efflux pumps. My research focuses on the identification and characterization of compounds with novel activity against C. albicans. Leveraging the Boston University Center for Molecular Discovery (BU-CMD)’s chemical library, I screened 3,049 compounds against a C. albicans clinical isolate and identified 17 molecules that inhibit C. albicans growth through metal chelation. Media supplementation with ferric or ferrous iron rescued C. albicans growth, indicating these compounds exert their antifungal activity primarily through iron chelation. Furthermore, I characterized the mode of action of two compounds with novel antifungal activity from the Broad Institute Diversity-Oriented Synthesis (DOS) library. Through genetic approaches, I identified one molecule as an inhibitor of Erg11, despite the compound’s lack of a canonical azole ring, and another compound as a translation inhibitor. Future work will leverage biochemical methods to further define compound mode of action and focus on investigating the therapeutic potential of prioritized molecules. Overall, my research has identified compounds with novel antifungal activity that may have potential for much needed, future drug development.
Abstract #13
Targeted Gene Correction for Duchenne Muscular Dystrophy Using CRISPR/Cas9: Systemic Treatment and Phenotypic Improvements in Mice
Sina Fatehi, The Hospital for Sick Children
Duchenne muscular dystrophy (DMD) is the most common pediatric muscular dystrophy, with an incidence rate of 1 in 5,000 males due to its recessive X-linked mode of inheritance. Multi-exonic deletions that disrupt the open reading frame of the gene and stop dystrophin synthesis are the most common cause of the condition. The absence of dystrophin causes a buildup of damage in muscle cells during periods of contraction and relaxation. A curative therapy for DMD deletions is within reach due to advances in CRISPR/Cas genome editing. We propose a novel strategy using these systems to correct the DMD patient deletion of exons 52-54 through targeted DNA knock-in of the deleted exons. This system was initially tested in-vitro on mouse myotubes harbouring the Dmd Δ52-54 deletion followed by transition to a mouse model harbouring the same mutation. In both models we have observed significant restoration of the wildtype transcript and dystrophin positive fibres. The results of this project will demonstrate the efficiency of this gene editing strategy and determine its therapeutic capacity.
Abstract #14
Long-Term In Vivo Biocompatibility of a Hyaluronan-Based Vitreous Substitute
Adam Forman, University of Toronto Faculty of Engineering
Various vitreoretinal conditions, including retinal detachment, require a vitrectomy: surgical removal of the eye's vitreous humor to access and repair the retina. The vitreous is then replaced with a substitute material to tamponade the retina during recovery. Current clinical substitutes – namely gases and silicone oils – carry major drawbacks during patient recovery, which typically include blurred vision, extensive head posturing, high ocular pressure, and follow-up procedures. As a result, patients are precluded from work, leisure activities, and air travel for several weeks or longer. There is an unmet need for a new biomimetic vitreous substitute to improve patient recovery following retinal surgery. We developed a novel hydrogel vitreous substitute composed of hyaluronan (HA), a component of the native vitreous, crosslinked with poly(ethylene glycol) (PEG) using stable oxime chemistry. Our hydrogel is injectable, biodegradable, biomimetic, transparent, and non-swelling. In vivo biocompatibility of our vitreous substitute was demonstrated in New Zealand White (NZW) rabbits over a 180-day period through histological analysis of the retina and by monitoring intraocular pressure (IOP). In addition, electroretinography (ERG) measurements showed normal retinal function in Dutch-Belted Pigmented (DBP) rabbits that received the vitreous substitute. These results demonstrate that our novel hydrogel is a promising candidate as a vitreous substitute for patients undergoing vitreoretinal surgery.
Abstract #15
Trends in platelet count among ovarian cancer patients
Vasily Giannakeas, Lightening Talk Speaker, Women's College Hospital
Background: Ovarian cancer patients often present with an elevated platelet count (thrombocytosis) at the time of diagnosis. An elevated platelet count is associated with poor cancer-specific survival among these patients. It is unclear if there are trends in platelet count prior to an ovarian cancer diagnosis, and if platelet count differs among patients that survive versus patients that succumb to their disease.
Methods: Using electronic medical records in Ontario we identified provincial residents diagnosed with ovarian cancer between January 2007 and December 2015. We selected patients with one or more complete blood count (CBC) records in the two-year period preceding or following their cancer diagnosis. We divided the cohort into survivors and non-survivors based on a patient’s three-year cancer-specific survival status. Using biweekly intervals, we plotted the median platelet count in the two years before and after diagnosis, stratified by survivor status.
Results: Our cohort consisted of 6,451 ovarian cancer patients with an average age of 62.2 at diagnosis. In total there were 77,657 CBC records in the four-year observation period; the average number of CBC records per patient was 2.0 in the two years prior to diagnosis, and 10.0 in the two years after diagnosis. In the 24 to 6 months preceding the diagnosis platelet count rose slowly, followed by a rapid rise in the six months prior to diagnosis. At diagnosis, patients who died from ovarian cancer had a higher platelet count than patients who survived. After diagnosis, platelet count for both groups dropped precipitously to below pre-diagnostic levels by 24 weeks. From week 24 to week 48 platelet count rose again - but the rise was steeper for those who died than for those who survived.
Conclusions: Platelet count increases in the two years prior to an ovarian cancer diagnosis and is higher among patients with poor survival. Platelet count may serve as a biomarker for the presence of ovarian cancer.
Abstract #16
Systematic Discovery of RNA Binding Proteins that Regulate MicroRNA Secretion and Cellular Retention
Wenyuan He, University of Toronto Faculty of Medicine
Cellular microRNAs (miRNAs) can be selectively secreted or retained, adding another layer to their critical role in regulating human health and disease. Given the short length of miRNAs (approximately 22 nucleotides), their sorting may depend primarily on the sorting of interacting molecules, such as RNA-binding proteins (RNABPs), which have complex localization signals. Several RNABPs have been shown to interact with specific miRNAs for their localization, but a systematic approach is lacking. This study aims to define the overarching significance of RNABPs in the sorting of miRNAs, using experimentally determined binding motifs of 93 RBPs. Microarray was used to profile intracellular and small extracellular vesicles (sEVs) miRNAs of mHypoE-46 neurons. Statistical enrichment analysis revealed that miRNAs preferentially secreted into sEVs are enriched with motifs of RNABPs present in neuronal sEVs or known to regulate miRNA secretion, such as hnRNPA2B1. Conversely, miRNAs that predominantly remain intracellular feature motifs of RNABPs typically found within the cell. Additionally, multiple miRNA motifs associated with either preferential secretion or retention, as identified by Garcia-Martin et al. in Nature 2019, showed significant correlations with the motifs of the RNABPs present in the study. Based on the presence or absence of specific RNABP motifs, several simple machine learning models have achieved over 80% accuracy in predicting whether a miRNA will be preferentially secreted or retained. These findings suggest that certain specific and novel RNABPs play a significant role in the secretion and retention of miRNAs. For future miRNA therapeutic development, considering these interactions could be crucial to maximize delivery effectiveness and minimize off-target effects. This is PRiME related but not funded by PRiME.
Abstract #17
An injectable hyaluronan-based hydrogel for the controlled release of antisFRP2 and FcNoggin via fragment crystallizable domain (Fc) affinity peptides
Daniela Isaacs-Bernal, PRiME Fellow 2024, University of Toronto Faculty of Engineering
Antibody therapy offers a targeted and precise approach to treating prevalent degenerative diseases affecting the retina; yet long-term delivery strategies are required to minimize repetitive dosing and improve therapeutic outcomes. In most of the cases, repetitive bolus injections are needed to achieve a therapeutic benefit; however, these are associated with a myriad of undesired effects including cataracts, endophthalmitis, among others. Sustained release strategies for antibody therapeutics aim to overcome these hurdles by maintaining therapeutic concentrations while using fewer injections. Here, we engineered a novel hydrogel system for the controlled release of antibodies via affinity-mediated interactions with specific binding ligands. As a proof-of-concept, we formulated an injectable hydrogel comprising Fragment crystallizable domain (Fc) peptide ligands to control and sustain the release of anti-sFRP2 and Fc-noggin, both shown to be required to stimulate vision repair. First, we synthesized and modified two peptides with demonstrated binding to the Fc-domain of IgG1 (FcL1 and FcL2) and determined their binding strengths to antisFRP2 and FcNoggin via biolayer interferometry. We showed that while FcL1 binds both antisFRP2 and FcNoggin, FcL2 demonstrated binding to antisFRP2 but not to FcNoggin. We then immobilized FcL1 within a hydrogel vehicle to prove sustained release of both biologics. We designed a hyaluronan-based hydrogel that takes advantage of oxime reaction for network crosslinking and IEDDA chemistry for FcL immobilization. We confirmed that sustained release of bioactive antisFRP2 and FcNoggin was achieved in the presence of FcL1. The beauty of this process is that no modification or treatment to the antibody is required to control release. Instead of using harsh environmental conditions during the encapsulation process, the antibody and delivery vehicle are simply mixed together, and release is controlled by the inherent affinity between the antibody and Fc-ligand (FcL). In particular, using this platform technology for the sustained release of antisFRP2 and FcNoggin holds great promise to repopulate the damaged retina in a precise and controlled manner.
Abstract #18
Ultra-sensitive, point-of-need, miRNA profiling using CRISPR-Cas12
Idorenyin IWE, PRiME Fellow 2024, University of Toronto Leslie Dan Faculty of Pharmacy
Here, we introduce a novel approach for miRNA profiling at the point-of-need for severe sepsis, leveraging both novel and established biomarkers. This method employs CRISPR-Cas12, traditionally utilized for DNA detection, to identify miRNAs. The motivation for this project arises from the critical need for sensitive detection of miRNA biomarkers for sepsis.
At the foundation of our project is a new method that enables Cas12 to detect RNA, including short miRNA without the need for PAM sites. Historically, Cas13 was the go-to tool for such tasks, but the protein is susceptible to degradation, limiting its practical value. In contrast, Cas12 has remarkable stability and superior thermal resilience, making it well suited for diagnostics, particularly for samples with contaminants (salts, pH changes, etc.). There is also evidence that Cas12 has better specificity in comparison with Cas13, and additionally, Cas12 is widely available from commercial suppliers.
The global sepsis crisis, responsible for 11 million deaths each year, is worsened by delayed treatments and the lack of early detection biomarkers. Drs. Claudia dos Santos and Gilbert Walker have discovered specific miRNAs linked to sepsis, notably in Covid-19 patients at different stages of infection. Our goal is to harness our Cas12 platform to detect crucial sepsis miRNA biomarkers, such as miRNA-A and miRNA-B, facilitating timely and precise interventions. As an initial step, we have successfully demonstrated the Cas12 system's efficacy with miRNA-21, with detection in as little as 10 minutes. These promising results have prompted us to broaden our efforts to include a wider range of sepsis biomarkers.
Our current efforts are concentrated on developing the miRNA amplification components of our project, which will be integrated with the Cas12 platform to guarantee sensitive miRNA detection and profiling. Following this, the comprehensive system will undergo evaluation with contrived miRNA samples prior to testing with patient samples. This includes plans for implementation at the point of care at St. Michaels Hospital. Our vision is to provide a new tool in the toolbox of ICU physician in the triage of Covid-19 patients, ensuring the timely administration of the appropriate medication to the right individual.
Abstract #19
High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting CAF-induced cancer cell invasion in HNSCC
Kunal Karve, University Health Network (UHN)
Cancer metastasis is a complex cascade that involves activation of cancer cell invasion and migration. This activation is greatly attributed by multiple factors, including the tumor-microenvironment (TME). Cancer-associated fibroblasts (CAF), a prominent cell type within the TME, have been shown to promote cancer cell invasion and migration, causing metastasis in various cancers including head and neck squamous cell carcinoma (HNSCC). The molecular mechanisms of how CAFs promote HNSCC invasion remain elusive. Our research goal is to understand how CAFs influence HNSCC cells to become invasive and potentially metastatic. Using a top-down research approach, our aim is to identify novel therapeutic chemical probe / drug regimens that potentially target CAF-dependent HNSCC cancer cell-invasion. In-order to perform high-throughput small molecule screens, we have established a 384-well format, three-dimensional (3D) spheroid invasion assay, as a powerful tool to study CAF-dependent HNSCC cancer cell invasion. This platform is currently being used to screen small molecule libraries and identify putative molecular targets, providing insights into underlying mechanisms of CAF-induced cancer cell invasion and candidate therapeutic strategies.
Abstract #20
Development of PET Theranostics for Head and Neck Cancer
Meysam Khosravifarsani, Lightening Talk Speaker, University of Toronto Leslie Dan Faculty of Pharmacy
This study explores a PET theranostic approach to diagnose and treat head and neck squamous cell carcinoma (HNSCC). HNSCC refers to cancers of the oral cavity, larynx, pharynx, and esophagus, which cause around 9,000 new cases and 4,000 deaths annually in Canada (1). Local recurrence occurs in 40-60% of patients (2) and 20-30% of early-stage patients progress to metastatic HNSCC (3). Recurrent and metastatic diseases are difficult to treat, and patients have poor outcomes. To address this challenge, we are developing theranostic monoclonal antibody (mAb) and/or peptide radioligands labeled with positron or beta-particle-emitting radionuclides for PET imaging and targeted radiotherapy of HNSCC. These agents consist of 64Cu-labeled panitumumab F(ab')2 to visualize HNSCC recurrence or metastasis by PET, and 177Lu-labeled panitumumab F(ab')2 for treatment. Panitumumab is a clinically approved mAb that targets the EGFR overexpressed on more than 90% of HNSCC (4). We are further interested in developing peptide radioligands targeted to EGFR. One candidate is a cyclic version of the GE11 peptide [cGE11] which has been reported to target EGFR. A PET scan and biodistribution study results for 64Cu-panitumumab F(ab')2 in mice with s.c. human patient-derived xenografts (PDX), showed tumour uptake of 13.4±2.5 percent injected dose per gram (%ID/g) at 24h h post-injection but low normal tissue uptake, e.g. muscle (1.3±0.7 %ID/g). This provided high-quality PET image contrast for tumour vs. other tissues. Further, through molecular docking (MD) studies, the interaction sites of FITC-labeled cGE11 were compared to those of EGF. We then conducted an in vitro study of the binding of FITC-cGE11 to tumour cells that express EGFR, both in the presence and absence of excess EGF by flow cytometry. The results indicated that the binding of cGE11 was not competed by EGF suggesting it may bind to a different domain on the EGFR or exhibit non-specific binding. We are now developing alternative analogs of cGE-11 to improve their specificity for the EGFR. Nonetheless, our study found that 64Cu-panitumumab F(ab')2 is a promising radioligand that visualizes HNSCC tumors. In the future, we plan to complex panitumumab F(ab')2 to 177Lu and study this agent for treatment of HNSCC PDX. This project is being carried out as part of the PRiME-UHN clinical catalyst program. References: 1. Brenner DR, et al. CMAJ. 2020;192:E199-E205. 2. Adelstein DJ, et al. J Clin Oncol. 2003;21:92-8. 3. Pisani P, et al. Acta Otorhinolaryngol Ital. 2020;40: S1-S86. 4. Zimmermann M, et al. Radiat Oncol. 2006;1:11. Supported by a PRiME-UHN clinical catalyst fellowship and a NFRF grant.
Abstract #21
Identification of guide proteins: tweaking journey of membrane protein in a proximity-dependent manner
Jiyoon Kim, PRiME Fellow 2024, University of Toronto Faculty of Medicine
Membrane proteins are essential conductors of cellular harmony, orchestrating functions vital for cell signaling, transport, and enzymatic activity. Their precise positioning within the cell is not only critical for normal physiological processes but also a key player in the symphony of diseases that arise when their localization goes awry. Imagine this cellular landscape as a map, with proteins journeying to specific destinations. We aim to tweak this journey using a metaphorical GPS, introducing "guide proteins" to potentially reroute and redefine the destination of target membrane proteins. Employing advanced techniques like induced proximity screening, we seek to survey and pinpoint guide proteins capable of altering the localization of target receptors in a proximity-dependent manner.
Abstract #22
Unlocking the potential of mesenchymal stromal cells using combinatorial analysis to investigate the effects of donor heterogeneity and critical processing parameters on MSC critical quality attributes.
Oreoluwa Kolade, University of Toronto Faculty of Engineering
Background & Aim:
Mesenchymal Stromal Cells (MSCs), known for their inflammation-regulating properties, face variability challenges hindering clinical and commercial success. Employing Design of Experiments (DoE) and desirability analysis, this study investigates donor heterogeneity and Critical Processing Parameters (CPPs) used to manufacture MSCs and assesses their dual impact on potency and expansion. We evaluate the inter-connectedness of donors and CPPs in impacting MSC expansion and critical quality attributes (CQAs) using a robust statistical approach.
Methods, Results & Conclusion:
We use DoE to assess input parameters—donor heterogeneity, first-line CPP parameters including plating MSC density, medium composition, and oxygen concentrations—across 13 bone marrow donors, including osteoarthritis (OA) patients from a previous clinical trial (NCT02351011). Exploring interaction effects between donors and CPPs, an 8-gene curated panel of anti-inflammatory/angiogenic genes under licensed conditions serves as MSC CQAs. Using the DoE, we evaluate novel effects of donor heterogeneity and CPP conditions on MSC expansion and MSC CQAs.
Principal component analysis (PCA) unveils heterogeneity among 12 CPP combinations and 8 genes across 13 donors. Significant PC1 scores (p<0.01 for responders vs. healthy, p<0.0001 for responders vs. non-responders) emphasize donor's role in MSC heterogeneous potency. Conversely, CPP conditions drive MSC expansion, with human platelet lysate(hPL) or animal component-free (ACF)-supplemented CPPs exhibiting significantly higher cell expansion (p<0.001) than fetal bovine serum (FBS). The 8-gene panel was ranked using sensitivity analysis and identified TSG-6 (TNF-stimulated gene 6 protein), VEGF (Vascular endothelial growth factor), and PD-L2 (Programmed cell death 1 ligand 2) as most sensitive to variations in donor heterogeneity (CV ≥ 10%). Using individual gene CV as weights, MSC donors are ranked based on calculated desirability scores across 12 CPP combinations with an experimentally determined desirability threshold. Our rankings correlate with clinical data stratifying MSC donors based on responder and non-responder status using stringent pain and function thresholds. Our analyses pinpoint CPP conditions (30% of CPPs) resulting in both expanded MSC and MSC CQAs.
In summary, PC1 scores confirm donor’s role in driving MSC potency, while hPL or ACF-supplemented CPPs drive MSC expansion. Desirability scores inform optimal CPP selection for enhanced expansion and gene potency.
Abstract #23
Development of a hydrogel for local and sustained delivery of Insulin-like growth factor 1 to treat retinal degeneration.
Jonathan Labriola, University of Toronto Faculty of Engineering
Dry-Age Related Macular Degeneration (dry-AMD) is the leading cause of blindness among the elderly. One of the hallmarks of this disease is chronic oxidative stress and inflammation in the outer layers of the retina. This leads first to death of the retinal pigmented epithelial (RPE) cells, followed by atrophy of the light sensing photoreceptor cells, ultimately causing blindness. Insulin like growth factor 1 (IGF1) is a pleiotropic growth hormone with demonstrated neuroprotective and anti-inflammatory effects, making it an intriguing therapy to treat dry-AMD. Despite this, IGF1 faces hurdles in clinical translation due to poor pharmacokinetics and side-effects with systemic delivery. Sustained local delivery of IGF1 is a potential solution to these issues. Presented here is a novel hydrogel that is ideal for injection into the eye which has been engineered to stabilize and sustain release of IGF1 for over a week. This sustained IGF1-release hydrogel (I-SR hydrogel) was tested in the rd10 mouse model of retinal degeneration. Mice treated with I-SR hydrogel for a week showed preservation of the photoreceptor layers in their retinas compared to mice treated with IGF1 in saline or saline alone. The next steps are to test functional and behavioural effects of I-SR hydrogel in rd10 mice.
Abstract #24
Cellspot: a digital microfluidic CHO cell testing platform
Jeremy Lant, University of Toronto Faculty of Arts and Science
In recent years, monoclonal antibodies (mAbs) have become a dominant force in the treatment of human diseases. Currently, over 100 mAbs have received approval for therapeutic use in the US, with wide-ranging applications from cancer to infectious diseases. The predominant method of producing antibodies for therapeutics involves expression in Chinese hamster ovary (CHO) cells. In the mAb production process, significant optimization is typically done to maximize antibody titres from CHO cells grown in bioreactors. Therefore, systems that can minimize and automate cell line testing (e.g., viability and antibody production assays) are valuable in reducing therapeutic mAb development costs. Here we present a novel platform for CHO cell line optimization using digital microfluidics. The system enables testing of CHO cell culture samples in 6-8 μL droplets with automated viability, media pH, and antibody production assays. This represents a significant reduction in sample volume requirements for CHO cell line testing, with potential for a fully automated micro-culturing platform. We anticipate that this technology and future iterations will help reduce the time-to-market and development costs of antibody-producing CHO cells.
This work is the product of a collaboration between the Wheeler lab (University of Toronto) and Sartorius Stedim North America Inc. (Cambridge, MA).
Abstract #25
Co-delivery of a Thermostabilized Enzyme and Human Neural Progenitor Cells Using Specialized Hydrogels Enhances Recovery after Stroke
Nitzan Letko Khait, Lightening Talk Speaker, University of Toronto Faculty of Engineering
Stroke is one of the leading causes of morbidity and long-term disability worldwide, with no clinically available treatments to promote tissue regeneration. Transplantation of neuronal cells is a promising approach, yet cell survival remains a challenge. To overcome these obstacles, we designed an injectable hyaluronan-based hydrogel that mimics the brain’s extracellular matrix and supports human induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) survival and neuronal differentiation. We encapsulated iPSC-NPCs in the hydrogel, injected it through a fine needle, and showed high cell viability and differentiation for at least 14 days in vitro.
To further enable cell survival and neuroplasticity, we engineered the bacterial enzyme chondroitinase ABC (ChASE) to have enhanced thermostability by introducing 37-point mutations (ChASE37). ChASE37, similarly to the native enzyme, degrades the inhibitory chondroitin sulfate proteoglycans (CSPGs) that are secreted after injury to the central nervous system. We expressed the enzyme as a fusion protein with a Src homology 3 (SH3) domain (SH3-ChASE37) and modified an injectable cross-linked methylcellulose hydrogel with SH3 binding peptides to enable affinity-controlled release. The hydrogel attenuated the release of bioactive SH3-ChASE37 in vitro and in vivo, and the efficacy of the released enzyme was demonstrated with degradation of the inhibitory CSPGs.
We co-delivered both iPSC-NPCs and affinity release SH3-ChASE37 directly to the brain of endothelin-1-induced stroke injured model, where we observed improved motor function. We are currently investigating the fate of the transplanted cells and the effects of SH3-ChASE37 on CSPG degradation and tissue plasticity.
Abstract #26
Investigating the potential weight loss and appetite suppressing effects of a plant phytohormone.
Calvin Lieu, University of Toronto Faculty of Medicine
Compound X (CX) is a plant phytohormone that controls differentiation, flowering, and cell division in plants. Recent studies have uncovered the effects that phytohormones could have in mammals, such as anti-inflammation and anti-aging. Preliminary work in our lab has found that CX could suppress the mRNA expression of the orexigen neuropeptide Y in hypothalamic neuronal models. Thus, we hypothesized that CX would suppress appetite in mice leading to weight loss and an improved metabolic state. To assess the effects of CX in mice, we fed male CD-1 mice a 60% high fat diet (HFD) for 4 weeks prior to daily oral feeding of a CX emulsion for 2 or 4 weeks while remaining on a 60% HFD. The mice exposed to CX ate significantly less than control mice and lost significant body weight. At the end of the exposure period, the mice underwent glucose tolerance testing and mice exposed to CX for 4 weeks had significantly lower fasting blood glucose levels. The hypothalamii collected from these mice after CX exposure displayed significant changes in hypothalamic feeding neuropeptide mRNA expression. Circulating metabolic hormone concentrations were quantified using a Luminex multiplex immunoassay and mice administered CX had significantly lower circulating levels of insulin and leptin. Our research establishes the ability of CX to induce body weight loss/maintenance and improve the metabolic state of HFD fed mice. Ultimately, these studies demonstrate the potential of a plant-based molecule in treating obesity.
Abstract #27
Discovery and Analysis of Microbiome-derived Molecules Targeting Nuclear Receptors
Jiabao Liu, University of Toronto Faculty of Medicine
The vast microbial community residing within the human gastrointestinal tract profoundly influences physiology, metabolism, and overall health. Microbial metabolites play a crucial role in shaping mood and behavior, while diseases such as metabolic, immune, and gastrointestinal disorders, with potential cancer implications, are intricately linked to these microbial activities.
Nuclear receptors (NRs), as ligand-binding transcription factors, serve as pivotal regulators, coordinating feeding behavior with systemic metabolic and immune functions. NRs also govern various temporal and behavioral processes, including developmental and circadian timing, sexual behavior, fertility, mood, and stress. NRs, at the intersection of diet, gut microbiome, and host, respond to the binding of small hydrophobic molecules, modulating subcellular localization, cofactor interactions, and transcriptional activities. Despite progress, a significant fraction of NRs lacks identified microbial ligands, underscoring the ongoing pursuit of the full spectrum of gut microbial ligands.
Employing an optimized Affinity Purification – Untargeted Mass Spectrometry (AP-UMS) approach using affinity-tagged constructs for human NR ligand binding domains, seven NRs underwent analysis against microbiome extracts. This effort led to the identification of masses corresponding to 26 new ligands, 14 of which were successfully validated and further assessed through in vitro and cell-based assays, with two currently under animal study. Given the success of translating NR ligands into therapeutics, these discoveries hold the promise of unlocking novel and safe biotic and probiotic interventions for disease management.
Abstract #28
Temporal and Palmitate-Mediated Regulation of MicroRNAs during Differentiation of IPSCS to Hypothalamic Neurons
Neruja Loganathan, University of Toronto Faculty of Medicine
Control of hypothalamic development and disease, including obesity, involves microRNAs (miRNAs) that negatively target mRNAs. These miRNAs have the potential to be used as biomarkers and therapeutics in disease. However, having been primarily studied in rodent models, the function of these miRNAs in the human hypothalamus remains unknown. Hypothalamic cells derived from human induced pluripotent stem cells (iPSCs) have recently enabled the study of the human hypothalamus at the cellular level. We found that expression of Dicer1, a key miRNA biosynthesis enzyme, increases during differentiation of iPSCs to hypothalamic cells. Therefore, we hypothesized that the function and targets of specific human miRNAs that are temporally regulated by differentiation can be studied using this model. To obtain a candidate list of miRNAs related to obesity and hypothalamic function, we performed a microarray with hypothalamic samples from CD1 mice fed a 60% high fat diet (HFD) for 6 weeks. Of the 23 upregulated and 36 downregulated miRNAs, we analyzed the top 8 miRNAs ranked by expression level in the human brain using qPCR. mir-30a-5p, mir-543, mir-10a-5p, mir-33a-5p, mir-33b-5p, mir-1-3p, mir-484 were upregulated, while miR-150-5p expression was downregulated throughout differentiation. Differentiated cells were then treated with palmitate to compare fatty acid-induced changes in the human model with the HFD-fed mice. Only miR-150-5p, miR-33b-5p and miR-484 were upregulated after exposure to 50 uM palmitate for 24 h. We are now investigating the molecular targets of these miRNAs and whether chronic palmitate exposure alters the miRNA profile and differentiation of hypothalamic cells. Ultimately, this study aims to further characterize iPSC-derived hypothalamic cells as models to study human hypothalamic disease.
Abstract #29
Towards in-vivo visualization of tRNA using RNA engineering
Colette Macarios, University of Toronto Faculty of Arts and Science
Transfer RNAs (tRNAs) are a key substrate for aminoacyl-tRNA synthetases which utilize ATP to load tRNA with an activated amino acid. The charged tRNA is then used by the ribosomes to facilitate proper protein synthesis. Although tRNA dysregulation has been associated with neurological diseases, there is little understanding about tRNA half-life, tRNA localization, and how tRNA is trafficked within mammalian cells. In this study, we engineered tRNAs to be fluorescent to enable live cell imaging and aim to track processes such as tRNA regulatory mechanisms as they occur within living cells. We designed several mammalian arginine tRNA constructs which are fused with a fluorescent aptamer (Mango II) at positions that should not interfere with their function. We could confirm aminoacylation of the engineered tRNA by the corresponding cognate aminoacyl-tRNA synthetase through ATP consumption assays. At present, we are introducing the tRNA fusion construct into HEK293T cells to determine whether the engineered tRNA would support endogenous protein synthesis. By engineering tRNAs to be fluorescent in vivo in mammalian cells, we are developing a visualization tool that can track tRNA trafficking and provide insight into where tRNA dysregulation and improper protein synthesis may occur. Visualizing disrupted tRNA processes in vivo could lead to a new understanding of different disease mechanisms.
Abstract #30
Characterization of a novel I450N gain-of-function mutation in the SH2 domain of the cytoplasmic tyrosine kinase JAK1
Pranshu Maini, The Hospital for Sick Children
Authors: Pranshu Maini1,3, Vritika Batura1,6, Bronwynn Simpson1,4, Amogh Natu1,7, Conghui Guo1, Lorraine Stallard4, Neil Warner1, Aleixo M. Muise1,2,6,7*
Affiliations: 1SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada, 2Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, Toronto, ON, Canada, 3Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada, 4Faculty of Arts and Science, Queen’s University, Kingston, ON, Canada, 6Department of Biochemistry, University of Toronto, Toronto, ON, Canada, 7Institute of Medical Science, University of Toronto, Toronto, ON, Canada,
Background: Inflammatory Bowel Disease (IBD) is a chronic illness characterized by debilitating inflammation of the gastrointestinal tract that affects 1 in every 140 Canadians. While IBD is typically considered to be a complex polygenic disorder, a diverse spectrum of rare genetic variants can cause monogenic forms of IBD. Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling mediates many known immune-regulatory pathways and is dysregulated in a variety of inflammatory diseases. Gain-of-function (GOF) mutations in the regulatory pseudokinase domain of JAK1 have been previously described in patients with immune dysregulation, which were successfully treated with JAK inhibitors. Jakafi® (Ruxolitinib), Rinvoq® (Upadacitinib), and Xeljanz® (Tofacitinib) are three such drugs that have been recently approved for the treatment of IBD, and are known to lead the rapid resolution of clinical disease.
Methods: Next generation DNA sequencing identified a rare and potentially deleterious heterozygous mutation in a highly conserved residue that maps to the regulatory SH2 domain of JAK1 in a pediatric IBD case dominantly inherited from the affected mother. The variant was validated using Sanger sequencing. Phospho-specific antibodies recognizing activated JAK1 and its downstream substrates, STAT1 and STAT3, were used in western blot experiments to investigate the activity of the JAK/STAT signaling pathway in primary patient-derived cells. Additionally, bulk RNAseq based transcriptional profiling of patient blood was used to examine altered gene expression.
Results: Significant phenotypic overlap was observed between our patient and published JAK1 GOF cases. Peripheral blood mononuclear cells (PBMCs) from our proband and affected mother showed elevated STAT1 phosphorylation under basal conditions compared to a healthy control. Furthermore, activation of the JAK1/STAT1 and JAK1/STAT3 signaling pathways in a patient-derived lymphoblast cell line using interferon gamma (IFNɣ) and interferon alpha (IFNα), resulted in hyperphosphorylation of STAT1 and STAT3, respectively. Moreover, patient-derived lymphoblasts treated with varying doses of JAK inhibitors, Ruxolitinib or Tofacitinib, notably inhibited STAT phosphorylation in a dose-dependent manner.
Conclusions: Our study supports JAK1 I450N as being a GOF variant. These results also suggest the therapeutic potential of targeting JAK1 kinase activity as a promising precision medicine-based strategy for the treatment of IBD in affected members of this family.
Abstract #31
Distinct endothelial cell populations characterize the pulmonary endarterectomy specimen in chronic thromboembolic pulmonary hypertension
H.S. Jeffrey Man, Lightening Talk Speaker
Authors: H.S.J. Man, Y.D. Zhao, L. Wu, U.Asghar, M. de Perrot
Purpose: Misguided vascular repair is thought to contribute to the pathophysiology of chronic thromboembolic pulmonary hypertension (CTEPH), yet little is known about endothelial cell (EC) phenotypes in the organized thromboembolic material of patients with CTEPH. Therefore, we sought to characterize ECs from pulmonary endarterectomy (PEA) specimens.
Methods: Human PEA specimens from proximal pulmonary arteries were collected from five CTEPH patients and dissociated for droplet-based single-cell RNA sequencing. Data from three control human lungs was obtained from the Human Lung Cell Atlas (https://hlca.ds.czbiohub.org accessed Sept. 9, 2021).
Results: From the PEA specimens of five CTEPH patients, we obtained transcriptional profiles for 11068 cells representing 20 clusters and 9 cell types. From those, 4 clusters displayed markers of ECs. To identify appropriate control ECs, we assessed markers of endothelial subtype from the Human Lung Cell Atlas and identified markers of pulmonary and bronchial artery ECs. Therefore, we obtained the transcriptional profiles of pulmonary and bronchial artery ECs from the Human Lung Cell Atlas as controls. After standardizing quality control metrics, we merged the data from 2174 control and 1772 CTEPH ECs. Dimension reduction of the merged dataset revealed 12 endothelial clusters (Figure). We note disproportionate distribution of control vs CTEPH ECs across clusters, with a majority of clusters biased towards one condition. Between and within clusters, CTEPH ECs displayed greater expression of TGFβ1, a marker of endothelial-mesenchymal transition (EMT), and P-selectin, a marker of endothelial activation.
Conclusion: Significant endothelial heterogeneity exists in both control and CTEPH ECs. However, we find differences in the composition of EC phenotypes between these two groups. We identify marker genes for CTEPH ECs that contribute to our understanding of EC phenotypes and misguided vascular repair in CTEPH.
Abstract #32
CRISPR genome editing in ex vivo human lungs for organ-centric genetic medicine
Kumi Mesaki, PRiME Fellow 2024, University Health Network (UHN)
The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ-centric genome-targeting therapeutics. One particularly exciting biomedical application is genetic engineering to create less-immunogenic donor organs to broaden the patient base and improve post-transplant outcomes. We envisioned genetically modifying donor lungs prior to implantation using our ex vivo lung perfusion (EVLP) platform to deliver immunologically optimized donor organs to recipients. However, assessing genome-targeting interventions in a human-relevant manner prior to clinical trials remains a major challenge due to the limited modalities available. To overcome this translational barrier of CRISPR-based organ therapies, we explored a novel platform for testing genome editing in human lungs ex vivo, simulating pre-implantation genetic modification of donor organs.
We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene IL-10 and developed adenoviral vectors carrying editing enzyme and guide RNA along with human IL-10 cDNA (for early IL-10 expression prior to genome editing). Human lungs from two donors, which were declined for clinical transplantation with research consent, were perfused and ventilated using the Toronto EVLP system. We delivered the adenoviral vectors via direct administration to the organ through the airway on the EVLP platform and continued perfusion for 12 h at physiological conditions. The treated lungs were stably perfused and showed an increase in IL-10 protein in bronchial wash during 12 h of EVLP. Upon completion of the EVLP, thin tissue slices were generated using precision-cut tissue slice (PCLS) technology and cultured for 14 days. NGS detected significant editing in the tissue from the treated lungs on day 7 and day 14, demonstrating sustained editing in human lung tissue.
Collectively, our findings demonstrate the feasibility of testing genome-editing interventions in a first-in human-organ study, representing an innovative avenue for the safe and accelerated clinical translation of CRISPR therapeutics addressing a significant translational barrier.
Abstract #33
Identification of Phenotypic State Determinants in Glioblastoma Stem Cells
Fatemeh Molaei, University of Toronto Faculty of Medicine
Glioblastoma (GBM) is a common and highly lethal type of primary brain tumor in adults which is highly resistant to standard chemo-radio therapy. Therapeutic failure is partly attributed to a pre-existing fraction of Glioblastoma Stem Cells (GSCs) that show high levels of plasticity and heterogeneity. Recent studies have shown that GSCs can be divided into two major subtypes, developmental (Dev) and injury-response (IR) according to their gene expression profiles and functional vulnerabilities.
A growing body of evidence indicates that in response to treatment, Dev- GSCs can transit into IR-GSCs, which are more invasive and therapy resistant. However, it is remained to be determined what genes are required to maintain IR-GSCs, knowledge which will has potential therapeutic for improving GBM prognosis. To this end, the aim of my project is to identify and define IR state determinants by performing genetic screens.
We performed a FACS-based genome-wide CRISPR-Cas9 knockout (KO) screen on patient derived IR-GSC using the TKOv3 gRNA library to identify regulators of phenotypic state based on expression of the IR-specific cell surface marker CD44. A comparison of gRNA abundances in CD44-low (bottom 15%) and CD44 high (top 15%) GSC populations enabled the identification of genes involved in regulation of IR-GSC state.
Notably, knockouts (KO) of EP300, a histone acetyl transferase, was enriched in the CD44-low population. Transition between cell states requires epigenetic reprogramming driven by alterations in chromatin accessibility a process in which histone acetylation plays an important role. Therefore, we focused on validation and characterization of EP300. Importantly, RNA-seq on IR GSC with EP300 KO showed significant downregulation of the IR state gene expression signature in comparison to control cells. Furthermore, the sphere forming ability of IR GSCs was abrogated upon KO of EP300. Our results lead us to postulate reduction of EP300 activity could reduce plasticity and aggressiveness of GBM.
Abstract #34
Hydrogel-encapsulated polarized macrophages for osteoarthritis treatment
Shahrzad Nouri, Lightening Talk Speaker, University Health Network (UHN)
[This is a PRiME funded project]
Osteoarthritis (OA) is a common joint disease associated with constant inflammation. OA affects more than 4 million Canadians and is estimated to increase to 1 in 4 by 2040. Studies show that 80% of OA patients are in constant pain and fail to perform their daily activities, significantly affecting their quality of life. Despite extensive research efforts, there are still no treatments for OA. Monocytes and macrophages (MΦs), cells of the immune system, play a central role in controlling inflammation in OA; thus, targeting MΦs has become an increasingly popular approach to developing cell-based OA therapies. We identified a pro-resolving MΦs (M2-like) subtype that showed promising results in resolving tissue inflammation after being injected in the joint of the experimentally induced OA mice. However, the results were not always conclusive and were affected by different factors, including low stability and impaired functionality of MΦs after the injection. This work aims to optimize the efficiency of MΦs by polarizing them to have pro-resolving properties and encapsulating them in alginate-based hydrogels in a developed microfluid channel before injection into the joint. This method will help increase the survival of the MΦs by providing a controlled physiological 3D environment before and after injection. We showed that M2-like pro-resolving macrophages, polarized by cytokines and encapsulated in alginate microspheres, maintained their pro-resolving phenotype for 10 days in culture. In contrast, free cells lost their differentiation and polarization within 3 days. To investigate the cross-talk between various populations of human resident cells and infiltrating monocytes in the joint, we performed a series of co-culture experiments. Our ongoing observations revealed the more prolonged survival of the macrophages in the presence of joint fibroblasts. The results of this study will further provide insights into how the interaction of different cells contributes to the development of diverse inflammatory conditions in the joint and the progression of OA. The outcomes of this research will advance the application of pro-resolving macrophages in OA treatment, leveraging optimized cell-based delivery systems.
Abstract #35
Antibody Synthesis in Cell-Free for High-Throughput Production and Screening
Sabina Panfilov, University of Toronto Leslie Dan Faculty of Pharmacy
Background: The generation of antibodies is well established in cell-based platforms; however, these are time consuming and costly processes. As an alternative, cell-free protein synthesis (CFPS) systems have been used to express antibodies. Naturally antibodies are folded in periplasmic conditions in E.coli, which is eliminated when using CFPS. Therefore, replication of the periplasmic environment is required in order to express properly folded antibodies. Different additives at different concentration need to be optimized per each antibody of interest, which makes the process time consuming.
Objective: This project aims to develop a platform for expedited prototyping and screening of reaction conditions utilizing an E.coli-lysate-based CFPS system. This kind of platform can be further used to expedite the screening process of antibody libraries. The proposed platform is based on CFPS reactions set-up in a multi-well manner followed directly by western blot and ELISA assay to identify properly folded antibodies with target binding affinity and high yield. In addition, a linear DNA protection system, that was developed in the lab, is used to prevent DNA degradation.
Results: The conclusions from the preliminary experiments helped to define the necessary additives to support disulfide bond formation for proper antibody folding. The high throughput platform was tested for Trastuzumab antibodies and will be followed with antibody library screen. In addition, we have demonstrated expression of 38 antibodies against 6 different antigens. Furthermore, we have utilized our ability to express antibodies and demonstrated conjugation of fluorescent payloads to CFPS-antibodies which is being confirmed in an immunostaining assay.
Abstract #36
A high-throughput kinase inhibitor screen using a 3D human disease model of obese adipocytes
Vera Pieters, PRiME Fellow 2024, University of Toronto Faculty of Engineering
Obesity is a major risk factor for the development of comorbidities such as type 2 diabetes and cancer and is initiated by the enlargement and subsequent exhaustion of adipocytes. However, studying adipocytes in vitro in 2D is complicated by their large size, sphericity and buoyancy. To address this, we have developed a 3D cell culture platform of primary human obese adipocytes using robotic and automation strategies in a 384-well array. This platform can be employed for high-throughput image-based morphological screens to study adipocytes during development of obesity. To understand the dynamic range of an image-based screen, we will first generate a wide spectrum of extreme adipocyte morphological phenotypes using a selection of kinase inhibitors, as kinases are involved in major signal transduction pathways. We then aim to integrate this validated high-throughput screening platform with our established kinase inhibitor library to identify the role of kinases in the development of obese adipocytes. We will confirm hits by performing CRISPR knockouts of kinases using soluble Cas9 in these primary cells for which stable Cas9 expression is notoriously difficult to achieve. We anticipate that this work will identify kinases and major signal transduction pathways that regulate lipid storage in adipocytes which will contribute to the development of obesity treatments.
Abstract #37
Interpretable factor decomposition of single-cell transcriptomics map of the rat liver
Delaram Pouyabahar, University of Toronto Faculty of Medicine
Single-cell RNA sequencing technologies have equipped biologists with a powerful tool to profile heterogeneous cellular systems, including the human body, and study the variability in gene expression levels between cells. Improvements in cost, throughput, and accessibility of such technologies have led to the fast growth of single-cell genomics data, increasingly requiring powerful computational tools to analyze. One major data analysis challenge is that mapping the information obtained from different sources is confounded by many technical and biological factors. Factor analysis, a set of well-established methods in machine learning, is useful for unraveling variability among measured variables by representing them in terms of a smaller number of unobserved variables, or factors. I aim to develop factor analysis methods that effectively disentangle the biological and technical sources of variation within single-cell transcriptomic maps. In the project's first phase, I developed a varimax-PCA-based pipeline to remove technical contamination from a multi-strain map of the healthy rat liver, identifying strain-specific variations. I recently expanded this method into a scalable statistical framework for integrating diverse single-cell maps, accommodating both biological and technical heterogeneity. The next step involves applying this model to study rejected and tolerant liver single-cell RNA-seq atlases, aiming to identify variations between the two transplant models and uncover signals influencing the liver microenvironment towards graft acceptance. Our method serves as a robust and versatile framework for the comparative analysis of single-cell datasets. Its utility extends beyond liver transplant models, offering valuable insights into the major drivers of transcriptome variation across a wide spectrum of experimental and biomedical settings.
Abstract #38
Effects of Low-Magnitude High-Frequency Vibration on Prostate Cancer Progression and Bone Metastasis
Amel Sassi, University of Toronto Faculty of Engineering
INTRODUCTION: Prostate cancer preferentially metastasizes to bone, which typically leads to bone pain and fractures. Notably, 80% of men who die from prostate cancer exhibit signs of bone metastases. To mitigate these effects, exercise is often recommended to cancer patients due to the beneficial effects on bone remodeling. However, physical activity may be challenging for elderly or bedridden patients. As such, vibration has emerged as a safe, effective, and easy to perform alternative therapy. Specifically, low magnitude high frequency (LMHF) has been shown to activate osteocytes and thereby reduce breast cancer cell migration. Nevertheless, the effects of vibration on prostate cancer progression and extravasation remains to be elucidated. We hypothesize that LMHF vibration (0.3 g, 60 Hz, 1h/day) will decrease prostate cancer cell extravasation through the activation of osteocytes and LMHF will directly influence prostate cancer cell growth.
METHODS: The bone-metastasis-on-a-chip model was fabricated using the methods outlined in Mei et al. (2019). Osteocytes were seeded into the osteocyte channel. Endothelial cells and prostate cancer cells were seeded into the lumen channel. Microfluidic devices were placed on a custom-made vibration platform for 1 hour every day for 3 consecutive days. Extravasation distance for each of the side channels was quantified. To further examine how vibration stimulated osteocytes regulate PC3 extravasation, we examined the effects of PC3 adhesion onto HUVECs treated with either static or vibration-stimulated MLO-Y4 condition media (CM). Additionally, to assess the effects of vibration on prostate cancer cells, an apoptosis assay, viability, and colony formation assay was carried out.
RESULTS: Results indicate that LMHF vibration (0.3 g, 60 Hz, 1h/day for 3 days) significantly reduced extravasation distance by approximately 42.8%. We also observed that 29% more PC3 cells remained adhered to HUVECs in static MLO-Y4 CM when compared to vibration MLO-Y4 CM. Furthermore, no significant differences were observed in percentage of apoptotic cells and viability of prostate cancer cells treated with LMHF vibration. However, a significant reduction in colony formation was determined.
DISCUSSION: Results suggest that LMHF vibration may be effective at reducing the incidence of prostate cancer bone metastases through a reduction in extravasation and colony formation. Specifically, the reduction in extravasation may be due to soluble factors secreted by vibration stimulated osteocytes that decrease the adhesion of prostate cancer cells to the endothelial monolayer.
Abstract #39
Mechanical loading of osteocytes via oscillatory fluid flow regulates prostate cancer cell extravasation to bone in vitro
Kimberly Seaman, University of Toronto Faculty of Engineering
Bone metastasis occurs in the vast majority of advanced-stage prostate cancer patients. Exercise has been shown to attenuate metastatic tumour progression and preserve bone structure in recent in vivo studies. Previous in vitro studies have indicated that direct prostate cancer-osteocyte interactions under mechanical loading promote cancer cell growth and migration. However, these findings do not correlate with in vivo results, and are more reflective of late-stage colonization when the secondary tumour is in much closer proximity to the bone matrix. Therefore, the aim of this study is to elucidate the role of osteocytes during the initial stages of prostate cancer bone metastasis, specifically when cancer cells extravasate through the endothelial barrier before colonizing bone tissue. We hypothesize that osteocytes under mechanical loading may play an indirect role in early-stage prostate cancer bone metastasis by signalling to endothelial cells in the metastatic bone niche. Oscillatory fluid flow (OFF) was applied to osteocytes at a frequency of 1 Hz and a peak shear stress of 1 Pa for two hours in all experiments to simulate physiologically relevant flow experienced by osteocytes in vivo. Conditioned media collected from static or OFF-stimulated MLO-Y4 osteocyte-like cells were used to assess PC-3 prostate cancer cell adhesion and trans-endothelial migration. A well-established microfluidic tissue model capable of applying mechanical stimuli to osteocytes was used to assess PC-3 extravasation through a lumen lined with human umbilical vein endothelial cells (HUVECs). Adhesion and trans-endothelial migration assays indicated that mechanical loading of osteocytes reduced PC-3 adhesion HUVEC monolayers and trans-endothelial migration compared to static controls. Moreover, mechanical loading of MLO-Y4 cells and primary osteocytes extracted from 2-month-old male mice reduced both the extravasation distance and rate of PC-3 cancer cells in the microfluidic device. Application of a neutralizing vascular cellular adhesion molecule 1 antibody to HUVECs abolished the difference in PC-3 adhesion and extravasation rate between static and OFF-stimulated groups. Taken together, these findings will provide more information on the role of mechanical loading of osteocytes during the initial stages of prostate cancer bone metastasis, and aid in the development of osteocyte-targeted cancer mechanotherapies to improve patient outcomes.
Abstract #40
Screening proteins secreted by endothelial progenitor cells as mediators of liver X receptor-dependent anti-atherosclerotic activity
Sarah Shawky, University of Toronto Leslie Dan Faculty of Pharmacy
Sarah A. Shawky, Adil Rasheed, Michael F. Saikali, Rick Lu, Yangyushuang Xu, Ricky Tsai, Milica Radisic and Carolyn L. Cummins
Department of Pharmaceutical Sciences, Leslie L. Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada
Early atherosclerosis is characterized by endothelial dysfunction and monocyte-endothelial cell adhesion (MEA). The liver X receptors (LXRs) are known to be anti-atherogenic in part by promoting macrophage reverse cholesterol transport. However, LXRs have also been shown to mediate vasoreparation independent of cholesterol efflux gene expression, implicating additional cell types. We found that LXR activation by the synthetic ligand GW3965 in bone marrow-derived endothelial progenitor cells (EPCs) generates a secretome that decreases MEA in 2D culture and 3D organ-on-chip models, as well as aortic sinus lesion area in Ldlr-/- mice. Our goal is to identify and characterize protein(s) from the LXR-activated EPC secretome as potential therapeutic targets. Conditioned media (CM) was generated from EPCs treated in the presence of GW or vehicle. Human aortic endothelial cells (HAECs) were treated with GW- or vehicle-treated EPC CM, or one of 10 candidate recombinant proteins at their estimated concentrations in the GW-treated EPC CM before co-treatment with TNFα. HAECs were cocultured with labelled THP-1 monocytes to assess MEA or analyzed by RT-qPCR to measure the expression of inflammatory (Il1b, Il6), cell adhesion (Icam1, Vcam1, Sele) and cholesterol efflux (Abca1, Abcg1) genes. Four candidates (IL18-BP, CD5L, PPT1 and soluble TREM2 (sTREM2)) decreased MEA with comparable efficacy to the GW-treated EPC secretome alone. sTREM2 shows greatest promise as it decreases MEA dose-dependently and drives an anti-atherogenic gene expression profile in activated HAECs. Thus far, sTREM2 demonstrates the most promising anti-atherogenic potential. Next, we will assess its protective effects through AAV-mediated expression in an Ldlr-/- mouse model.
Abstract #41
Drug Response Prediction and Biomarker Discovery Using Multimodal Deep Learning
Farzan Taj, Ontario Institute for Cancer Research (OICR)
We present a novel algorithm, the Multi-Modal Drug Response Predictor (MMDRP), designed to accurately forecast how a specific cell line will respond to a small molecule cancer drug. This prediction is based on a combination of the drug's chemical structure and the cell line's 'omics' profiles, and can be used to potentially improve personalized cancer therapy.
We first highlight problems in pharmacogenomic efforts, such as data skewness, scarcity, and chemical compound representation. Our open-source Python-based program integrates various 'omics' data types and assigns higher weights to less frequently observed drug efficacies. It employs a graph neural network for drug representation, providing a more insightful representation of drug molecules and their structure. A final fusion method combines the complementary information from different 'omics' sources, enhancing the model's predictive performance.
We have rigorously evaluated our model using custom cross-validation schemes and demonstrated its performance across various scenarios in which different combinations of cell lines, drug scaffolds, and cancer types are available. To our knowledge, this is the first project assessing prediction performance across the range of drug efficacies rather than reporting a single measurement. Our work also provides a comprehensive assessment of the utility of different cell line profiling data types for DRP, demonstrating that combining multiple complementary 'omic profiles can improve drug response predictions. The methods employed in our work have addressed some of the existing challenges and have highlighted areas for future research to bring DRP closer to clinical use. The program is available at https://github.com/LincolnSteinLab/MMDRP.
Abstract #42
Ionizable Lipids Optimized for Intramuscular Delivery
Grayson Tilstra, PRiME Fellow 2024, University of Toronto Faculty of Engineering
Lipid nanoparticles (LNPs) are safe and potent delivery vehicles of mRNA. Following the success of COVID-19 mRNA vaccines developed by Moderna and Pfizer/BioNTech, intramuscular delivery of mRNA-LNPs is recognized as a clinically relevant route of administration. Of the different LNP components, the ionizable lipid is the main driver of efficacy making it an attractive target for molecular optimization. Despite many reports of chemically distinct ionizable lipids, the effect of the ionizable lipid structure on intramuscular mRNA delivery remains poorly understood. To address this knowledge gap, we synthesized a series of novel ionizable lipids and studied the effect of molecular structure on LNP physicochemical characteristics, intramuscular mRNA delivery, and pharmacokinetics
Small modifications of the ionizable lipid structure can drastically change the quality and efficacy of LNPs. Therefore, careful and intentional molecular design is required. Here, we report the design and optimization of a new family of ionizable lipids that comprise distinct regions (a tertiary amine core, a degradable linker, and hydrophobic tails). These three molecular regions were systematically altered to study their effects on LNP parameters, and in vivo expression and safety profile in mice.
This iterative screening strategy generated key structure-activity relationships: ionizable lipids that included hydrogen bonding groups and formed LNPs with an apparent pKa between 6.5 and 6.9 resulted in significantly higher intramuscular expression. Additionally, the in vivo clearance of ionizable lipids was dependent on the hydrolysis rate of the biodegradable linker. Our lead lipid achieved higher expression of an mRNA-encoded protein compared to industry standards DLin-MC3-DMA, SM-102 and ALC-0315. Optimization of the biodegradable linker resulted in rapid clearance from murine tissues after intramuscular administration.
Altogether, we discovered highly potent ionizable lipids and outline how the molecular structure of ionizable lipids affects LNP physical characteristics, mRNA delivery to the muscle, and tissue clearance. Our work provides design principles for ionizable lipids and lays the groundwork for safer and more effective LNPs for intramuscular administration.
Abstract #43
Metabolic Reprogramming and Resistance to BCL-2 Inhibition in Large Cell Lymphoma
Daisy Tran, University Health Network (UHN)
Large cell lymphoma is a B-cell malignancy in which the anti-apoptotic protein BCL-2 is often dysregulated by chromosomal translocations and/or overexpression. Lymphoma patients with BCL-2 dysregulation have particularly poor outcomes under standard chemoimmunotherapy treatment regimens and represent a clinical unmet need. BCL-2 sequesters the mitochondrial pore forming, pro-apoptotic, proteins BAX and BAK thereby preventing apoptosis and promoting cell survival. The discovery of the BCL-2 inhibitor, venetoclax, has provided an unprecedented opportunity to target BCL-2 in hematological malignancies. As with many cancer therapies, resistance and subsequent relapse remains an issue and resistance to BCL-2 inhibition is no different. Elucidating mechanisms of resistance is critical to forwarding treatment protocols by identifying potential routes for the development of combination therapies by dampening pathways that drive resistance or by targeting vulnerabilities that are associated with drug resistance.
To understand venetoclax resistance (VR), we created and characterized the phenotypes of VR models using lymphoma cell lines. We find that VR cell lines lose expression of BCL-2 entirely while upregulating the anti-apoptotic protein MCL-1. In addition, VR cells have altered bioenergetics as compared to their venetoclax sensitive (VS) counterparts. To further investigate the bioenergetic phenotype of the VR cell lines, we used a XFe96 bioanalyzer to measure real-time changes in cellular metabolism. We observed that VR cells had higher OCRs and extracellular acidification rates (ECAR) compared to the VS cell lines. Glycolytic ATP production rates in both VR and VS cell lines were greater than the mitochondrial ATP production rates with VR cells having a greater overall ATP production rate. Furthermore, VR cells have a more energetic phenotype that shifted from aerobic to glycolytic when under stress, while VS cells remained quiescent or lacked metabolic plasticity.
Taken together, we find that venetoclax has varying effects on the cellular metabolism of lymphoma cell lines. The findings in this study will guide subsequent investigations on the altered metabolic phenotype of VR cells and how they can be targeted to overcome mechanisms of venetoclax resistance and improve treatment options for patients with lymphoma.
Abstract #44
Identification of Selinexor-Modulated XPO1 Cargo in Diffuse Large B Cell Lymphoma
Kyla Trkulja, University Health Network (UHN)
Background: Exportin 1 (XPO1) facilitates the export of proteins from the nucleus to the cytoplasm of eukaryotic cells and is overexpressed in almost all cancers. Selinexor, a selective inhibitor of nuclear export (SINE) inhibits XPO1 and is approved as a treatment for relapsed/refractory diffuse large B cell lymphoma (DLBCL). However, critical knowledge gaps remain in understanding XPO1 biology and exported cargo, how they contribute to lymphoma, and the downstream consequences of XPO1 inhibition that lead to anti-cancer effects. These gaps are preventing selinexor from being used clinically in effective combination therapies. This research is being conducted to identify what cargo proteins are exported by XPO1 in DLBCL and how selinexor is able to modulate these processes. Identification of XPO1 cargo relevant to DLBCL will facilitate an improved understanding of selinexor’s mechanism of action in this disease to inspire effective combination therapies with the drug.
Methods: The initial steps for proximity-dependent biotinylation labelling (BioID) were performed on DLBCL cell lines. Cells were incubated with varying concentrations of selinexor or 3, 6, 12, and 24h and whole-cell lysates were subject to Western Blot to determine the effects of selinexor on XPO1 expression. Following this, lentivirus for gene delivery of miniTurbo, a mutant biotin ligase enzyme used for BioID, was synthesized and used to infect DLBCL cells. Infected and non-infected cells were supplemented with biotin for 15, 30, and 60 minutes. Whole cells lysates were obtained and subject to Western Blot to detect the presence of biotinylated proteins and successful gene expression.
Results: Selinexor affected XPO1 expression in a dose- and time-dependent manner, with the most significant trend being a dose-dependent decrease in XPO1 at 24h, suggesting an optimal dose of 0.5uM for future experiments. Detection of FLAG in the infected cells demonstrated successful gene expression, and these cells also showed a greater presence of biotinylated proteins when supplemented with biotin than non-infected cells, demonstrating activity of the system. Future work will complete the BioID assay by repeating these methods with the XPO1-miniTurbo fusion and identifying these biotinylated proteins by mass spectrometry analysis.
Abstract #45
Enhancing PDT efficacy at GBM infiltrates with a targeted nanoplatform using apoE mimetic peptide
Hanyi Weng, University of Toronto Faculty of Medicine
Glioblastoma (GBM) stands as the most prevalent primary brain cancer with a poor prognosis with a median survival rate of less than 15 months. 75-80% of patients have local recurrence after standard treatment due to GBM infiltration into parenchymal tissue. Photodynamic therapy (PDT) presents a promising avenue for treating GBM infiltration while preserving normal tissue, owing to its high safety profile. However, its efficacy is hampered by the heterogeneous accumulation of photosensitizers at GBM infiltrates resulting from the blood-brain barrier (BBB).
Apolipoprotein E3 (apoE3) has emerged as a potential targeted ligand for inducing transcytosis of nanoparticles across the BBB, facilitating active transport into GBM tumours through receptors present on both the BBB and those overexpressed on GBM cells. Despite its promise, the use of apoE3 encounters a translational barrier due to high costs relative to its low functional targeting domain. To address this, our study proposes the use of an apoE3 mimetic peptide to increase the functional targeting domains on nanoparticles. We hypothesize that this mimetic can form nanoparticles to enhance photosensitizer delivery in GBM cells across an intact BBB, ultimately boosting PDT efficacy.
Utilizing porphyrin as a model photosensitizer, we fabricated porphyrin nanoparticles with both full-length apoE3 and the mimetic peptide. We characterized the morphology, size, peptide assembly, and porphyrin loading of these nanoparticles, and our findings revealed that the mimetic nanoparticle maintained the structural characteristics of the full-length apoE3 nanoparticle. The mimetic nanoparticle exhibited a two-fold higher uptake in U87 GBM cells compared to apoE3 porphyrin nanoparticles. Ongoing experiments aim to validate the receptor-mediated uptake of mimetic nanoparticles and the BBB penetration abilities of the mimetic nanoparticle, with the anticipation of demonstrating its potential in improving porphyrin accumulation in GBM. We posit that this targeted approach holds promise for enhancing PDT efficacy in a more clinically translatable manner.
Abstract #45
Tumor-Tailored Ionizable Lipid Nanoparticles Facilitate IL-12 Circular RNA Delivery for Enhanced Lung Cancer Immunotherapy
Yue Xu, Lightening Talk Speaker, University of Toronto Leslie Dan Faculty of Pharmacy
Strategies employing lipid nanoparticles (LNPs) loaded with messenger RNA (mRNA) to regulate cytokine expression are promising in the field of cancer immunotherapy. Specifically, interleukin 12 (IL-12) is a cytokine of interest due to its potent anti-tumor effect. However, mRNA's transient expression due to its short half-life results in suboptimal pharmacokinetics compared to circular RNA (circRNA), which limits therapeutic efficacy. Simultaneously, the presence of tumor heterogeneity further increases the challenges of delivering RNA therapeutics to tumors via LNPs.
To address these, we develop an LNP platform tailored for IL-12 circRNA delivery in lung cancer models, demonstrating superior pharmacokinetic properties versus mRNA and lung cancer therapeutic effect. In the mouse lung cancer model, it remodels the tumor microenvironment, inducing potent anti-tumor immune responses. A single dose of IL-12 circRNA LNP triggers significant interferon-γ (IFN-γ) mediated T cell and natural killer (NK) cell activity, boosting immune cell infiltration in tumors. Moreover, intratracheal administration achieves significant tumor regression, mitigating adverse events associated with systemic administration. This customizable lipid nanoparticles platform for circRNA delivery demonstrates promise for sustained and potent cancer immunotherapy.
Abstract #46
Identification of optimized sequential multidrug cancer chemotherapies using the integration of patient-derived organoids grown in a microfluidic tumor-on-a-chip platform with machine learning
Ilya Yakavets, Lightening Talk Speaker, University of Toronto Faculty of Arts and Science
Ilya Yakavets a,#, Sina Kheiri b,#, Riley J. Hickman c,d, Faeze Rakhshani a, Jennifer Cruickshank e, Matteo Aldeghi c,d,f, Ella Rajaonson c,d, Edmond W.K. Young b,g, Alán Aspuru-Guzikc,d,f,h,i,j*, David W. Cescone* and Eugenia Kumacheva a,g,h,*
a Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, ON M5S 3H6, Canada
b Department of Mechanical & Industrial Engineering, University of Toronto, 5 King's College Road, Toronto, ON M5S 3G8, Canada
c Chemical Physics Theory Group, Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, ON M5S 3H6, Canada
d Department of Computer Science, University of Toronto, 40 Saint George Street, Toronto, ON M5S 2E4, Canada
e Princess Margaret Cancer Centre, University Health Network, 610 University Avenue, Toronto, ON M5G 2C1, Canada
f Vector Institute for Artificial Intelligence, 661 University Avenue, Toronto, ON M5G 1M1, Canada
g Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON M5S 3G9, Canada
h Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5, Canada
i Department of Materials Science & Engineering, University of Toronto, Toronto, ON M5S 3E4, Canada
j Lebovic Fellow, Canadian Institute for Advanced Research, Toronto, ON M5G 1Z8, Canada
Sequential combination chemotherapy can potentially offer great benefits for metastatic cancer treatment, however, the challenge is to identify the most effective regimens, that is, the time, doses, and drug administration sequence. This study introduces a novel approach to cancer therapy optimization by developing a time-sequencing platform that integrates machine learning (ML) with a tumor-on-a-chip microfluidics platform. We combined a cancer organoid model, microfluidics, and machine learning to discover new or optimize existing sequential combination chemotherapy. A microfluidic platform integrated with ML was used to grow up to 1200 organoids from breast cancer patients’ cells in the device with the size of a credit card and utilized for screening of the sequential administration of drugs. Based on the organoids’ response, the ML algorithm adaptively learns to propose drug combinations and administration regimens with higher and higher anticancer efficacy, thus, leading to the decision achieved in a time-effective manner. We validated the synergistic behaviour of a concurrent administration novel combination of Poly (ADP-ribose) polymerase inhibitors and Bromodomain and Extra-Terminal motif inhibitors on a triple-negative breast cancer organoid model. The ML-based Bayesian optimization algorithm integrated into the closed-loop workflow with microfluidic platform as the experiment planner was used to recommend next-step drug concentrations for administration and prioritize the most effective synergistic multidrug strategy. Utilizing the automated platform, we explored concurrent and sequential protocols of administration of drug combination and found that only concurrent administration of the drug combination yields synergistic efficacy. Results will inform the design of new clinical strategies, which will be tested in early-phase clinical trials at Princess Margaret Cancer Centre. The developed innovative translational approach represents a significant advance in the discovery and optimization of cancer treatment and improves the outcome for breast cancer patients in a highly personalized manner.
Abstract #47
Engineering Hyaluronan-Based Hydrogels to Deliver Endothelial Progenitors for Therapeutic Angiogenesis
Yuan Yao, University of Toronto Faculty of Engineering
Coronary artery disease is the leading cause of mortality worldwide. Therapeutic angiogenesis is a potential strategy to foster new vessel formation in the infarcted tissues and subsequently improve tissue remodeling and cardiac function following myocardial infarction. Human pluripotent stem cell (hPSC)-endothelial progenitors have shown ability to revascularize ischemic tissues, offering a unique opportunity to develop new therapies. However, their application potential is limited by low cell survival and low engraftment upon transplantation. To overcome these challenges, we designed an injectable hyaluronan (HA)-based hydrogel matrix as cell delivery vehicle. The hydrogel harnessed cell adhesion sites and promoted viability and angiogenic function of endothelial progenitors. Sodium hyaluronate of different molecular weights (10kDa, 40kDa, and 250kDa) were crosslinked using inverse electron-demand Diels–Alder chemistry. To enable the adhesion of endothelial progenitors, two cell-adhesive peptides, collagen-derived KGHRGF and laminin-derived NWRHSIYITRFG, were chemically immobilized to HA. To further promote cell adhesion, collagen was physically mixed in HA hydrogels. Viability and angiogenesis of hPSC-derived endothelial progenitors were assessed. We found that the viability and adhesion of endothelial progenitors were significantly affected by the molecular weight of HA. Cell viability drastically decreased as the molecular weight of HA increased to 250kDa. Cells on 40kDa HA exhibited highest viability and spreading. Further enhancement of cell adhesion and viability was achieved by increasing the collagen content in the 40kDa HA gel. Angiogenesis analysis demonstrated that the immobilized peptides stimulated the formation of tube-like structures by endothelial progenitors. Additionally, the hydrogel had an average gelation time of 5.1 min and remained injectable through during a 1-hour testing period. In conclusion, our novel formulation of HA-based hydrogels is capable of promoting viability and angiogenesis function of hPSC-derived endothelial progenitors, and is appealing candidate used for biomaterial delivered cell therapies to revascularize infarcted tissues.
Abstract #48
A Structure-Guided Approach to Identify Fungal-Selective Yck2 Inhibitors
Bonnie Yiu, University of Toronto Faculty of Medicine
Bonnie Yiu1, Emily Puumala1, Pascal Marchand2, Luke Whitesell1, Nicole Robbins1, Leah E. Cowen1
1Department of Molecular Genetics, University of Toronto, Canada.
2Cibles et médicaments des infections et de l'immunité, IICiMed, Nantes Université, France
Candida species are a major cause of invasive fungal infection with an associated mortality rate of ~64% despite treatment. The emergence of antimicrobial resistance coupled with the scarce antifungal arsenal highlights the need for novel therapeutics. Protein kinases have been rewarding targets in drug development for diverse diseases but remain untapped in the quest for new antifungals. This study builds on the success of a screen of kinase inhibitors with efficacy against a drug-resistant isolate of C. albicans, which revealed a 2,3-aryl-pyrazolopyridine molecule, termed GW, as an inhibitor of C. albicans yeast casein kinase 2 (Yck2). Yck2 is a fungal member of the casein kinase 1 (CK1) family, which is required for growth under physiological conditions, is important for maintaining echinocandin resistance, and plays a key role in virulence in a mouse model of infection. While GW demonstrates potent bioactivity against C. albicans, its poor metabolic stability presents a liability for the progression into in vivo studies. Therefore, we strived to optimize the GW scaffold based on insights obtained from co-crystal structures of Yck2 in complex with GW. These structural observations guided the generation of three sets of molecules: 1) bioisosters of GW employing an idazo[1,2-a]pyridine scaffold 2) R-substituents of the parent pyrazolo[1,5-a]pyridine, and 3) imidazo[1,2-x]azine derivatives. Utilizing genetic and biochemical approaches, I characterized nine newly-synthesized imidazo[1,2-x]azine derivatives for their potency and bioactivity against C. albicans. From these studies, two molecules, CTN1756 and CTN1844, were prioritized. Both demonstrated whole-cell bioactivity in a standard dose-response assay against C. albicans, and biochemical selectivity for fungal Yck2 compared to human CK1α. Furthermore, CTN1756 and CTN1844 displayed on-target whole-cell activity, as compound treatment results in polarized growth of C. albicans, and enhanced caspofungin efficacy in an echinocandin-resistant strain, consistent with Yck2 inhibition. Unfortunately, all imidazo[1,2-x]azine compounds tested to date exhibited low metabolic stability in vitro. Future work will focus on the generation of additional molecules to further optimize the potency, selectivity, and metabolic stability of Yck2 inhibitors as a novel class of antifungal.
Abstract #49
Human iPSC-derived platforms to model diseases of the central nervous system and screen for regenerative therapeutics
Nadia Zafar, PRiME Fellow 2024, University of Toronto Faculty of Medicine
Injury to the brain and spinal cord often results in poor functional recovery due to a lack of regenerative responses in the central nervous system. Although damage can occur through a variety of means, such as traumatic injury, stroke or neurodegenerative disease, the result is a complex cascade that ultimately permanently disrupts neuronal signaling via axons. Given that current therapies are ineffective in reversing damage, patients face drastically reduced life-expectancies, require long-term physical therapy and endure severe psychological, social and economic burdens. Thus, there is an imminent need for therapeutics that promote functional regeneration of affected neurons.
A challenge in developing regenerative therapies for the central nervous system is a lack of suitable human disease models. Stem-cell derived platforms represent a powerful tool to mimic human specific molecular signatures across various types of neurological diseases and applying these platforms towards drug screening can further provide insight into critical disease mechanisms that vary from patient to patient.
To this end, we will present the results of our preliminary efforts towards establishing human iPSC-derived platforms as models to mimic neurological disease and their use as a pre-clinical screening tool. Using human iPSC-derived neurons, we have implemented assays to model axonal damage and the inhibitory microenvironment. We have also used these assays to identify compounds that promote regeneration in damaged cortical neurons. The results of this work represent the utility of iPSC-derived platforms to investigate mechanisms and therapeutics for diseases affecting the central nervous system.
Abstract #50
Modulation of cell surface glycans and resulting effects on Calreticulin binding and phagocytosis
Logan Zettle, University of Toronto Faculty of Arts and Science
Calreticulin (CRT), an Endoplasmic Reticulum (ER) resident protein, can promote recognition and phagocytosis of cancer cells by macrophages when present on the surface of these cells. This surface localization may be due to translocation from the ER, or exogenously bound CRT secreted from macrophages. Recently, it has been discovered that removal of terminal N-acetylneuraminic acid (Neu5Ac) on malignancies reveals a cryptic tri-antennary/multivalent (Tri/Mii) glycan which binds secreted CRT, increasing phagocytosis.
In this work, we utilize sialyltransferase inhibitors to modulate surface Neu5Ac levels, leading to increased Tri/Mii moiety exposure, exogenous CRT binding, and phagocytosis. Tests were conducted using a range of hematopoietic cell lines, and pro-inflammatory macrophage-like cells were differentiated from the THP-1 monocyte cell line using phorbol 12-myristate 13-acetate and lipopolysaccharide. Recombinant CRT was fluorescently labelled and used to quantify surface binding. Surface glycan modulation and quantification of phagocytosis were conducted using flow cytometry. Surface CRT binding was successfully increased, and a resultant increase in phagocytosis was observed.
Abstract #51
Delineating the potent appetite suppressant effects of a novel plant hormone and its mechanism in defined hypothalamic neurons
Cindy Zhang, University of Toronto Faculty of Medicine
Obesity is primarily caused by a positive energy balance wherein energy intake exceeds expenditure. This balance is tightly regulated by the hypothalamus via the controlled release of feeding-related neuropeptides, including orexigenic neuropeptide Y (NPY) and agouti-related peptide (AgRP). Our lab recently discovered that a plant-based phytohormone, compound X (CX), decreases body weight and food intake in mice maintained on a 60% high-fat-diet-fed. Moreover, CX robustly reduces Npy mRNA expression in immortalized hypothalamic neurons as early as 4 hours after treatment. Given the importance of mTORC1 in feeding regulation, we hypothesized that CX suppresses appetite via inhibiting the central mTORC1 pathway. This study elucidates the CX-mediated mechanisms involved. RNA-sequencing was performed on an NPY/AgRP-expressing cell line, mHypoE-46, upon 4 or 16 hours of 100 μM CX exposure. Candidate pathways were selected via bioinformatic analysis using GSEA, ErmineR, and SwissTargetPrediction. The mTORC1 pathway was investigated due to its high ranking in all algorisms and key involvement in energy homeostasis. RT-qPCR validation demonstrated that sestrin2 (sesn2), an mTORC1 inhibitor, is significantly enriched by over 2-fold in CX-treated neurons. Concurrently, CX upregulated the expression of eukaryotic translation initiation factor 4E (4E-BP1) and unc-51-like autophagy activating kinase (ULK1), two direct effectors suppressed by mTORC1. Selective activators and inhibitors will be used to confirm the involvement of mTORC1 in CX-based control of energy homeostasis in hypothalamic neurons. Our study delineates the CX mechanism of action in the hypothalamic control of energy balance, providing fundamental insights into a novel, accessible obesity treatment for humans.
Abstract #52
A mineralized collagen-containing hydrogel for bone repair
Zi Xuan Zhang, University of Toronto Faculty of Engineering
Background: Recent advancements in repairing bone defects include the use of marrow-derived mesenchymal stromal cells (MSC(M)s) as an alternative to bone autografts. MSC(M)s can differentiate into osteoblasts that mineralize the extracellular matrix. However, the effectiveness of injecting cells alone is limited by poor retention at the defect site without an encapsulation material. Thus, there are increased efforts in engineering biomaterials, like injectable hydrogels, to address this challenge.
Objective/Hypothesis: A body of in vitro and in vivo evidence suggests that biomaterials mimetic of bone components (mineralized type I collagen) and substrate stiffness can inherently upregulate MSC(M) differentiation and bone mineral production. Therefore, I hypothesize that a biomimetic and mechanically stiff hydrogel containing mineralized type 1 collagen can accelerate bone repair by enhancing MSC(M) differentiation kinetics.
Methods: Lyophilized mineralized collagen, DMEM, and soluble collagen were combined to create hydrogels that form in situ upon injection into physiological conditions. To develop hydrogels with different stiffnesses, the amount of mineralized collagen, concentration of soluble collagen, and genipin crosslinking were varied. The stiffness was measured as the storage modulus using a rheometer. MSC(M) differentiation in vitro in the hydrogels will be assessed by qPCR and quantitative protein analyses. von Kossa assays and electron microscopy will be used to assess mineral nodule production.
Results: Increasing the concentration of soluble collagen from 3 to 4mg/mL improved the hydrogel stiffness by 3.5-fold. Control hydrogels without mineralized collagen have storage moduli between 150-200Pa. Mineralized collagen-containing hydrogels are significantly stiffer (p<0.0005) by 3-fold.
Conclusion: Preliminary rheology results show that the viscosity of soluble collagen and the incorporation of mineralized collagen both improve the stiffness of collagen-based hydrogels. The effect of varying genipin crosslinking on hydrogel stiffness and differentiation kinetics will be explored next.
Significance: My project entails the development of a hydrogel that is mimetic of native bone composition for progenitor cell differentiation and subsequent bone repair. The ability to tune the stiffness of the encapsulation system will broaden the applicability of current MSC(M) therapy.
Abstract #53
Exploring the Impact of MicroRNA Modulation on Macrophage-mediated Immune Regulation of Acute Respiratory Distress Syndrome (ARDS)
Nashmia Zia, Lightening Talk Speaker, University of Toronto Faculty of Arts and Science
Acute Respiratory Distress Syndrome (ARDS) presents a significant challenge in intensive care, characterized by high mortality and a lack of established therapies. MicroRNAs (miRNAs) show promise in alleviating alveolar dysfunction associated with ARDS. Key regulators of gene expression, miRNAs are implicated in modulating immune responses, particularly those involving macrophages in ARDS. This study aims to understand the immunomodulatory potential of miRNAs.
Methods: MiRNAs are loaded into lipid nanoparticles (LNPs) as a delivery system. In vitro screening techniques assess the impact of single and/or multiple miRNA modulations, with or without LNPs, on macrophage-mediated immune responses. A trans-well model, incorporating lung epithelial cells and human monocyte-derived macrophage is developed to mimic the physiological conditions of ARDS.
Results: Preliminary findings demonstrate that miRNA modulation can regulate capillary vascular leakage and inflammatory cell infiltration, impacting tight junction function between epithelial cells. The use of LNPs enhances the bioavailability and efficacy of miRNA delivery. In vitro screening of diverse miRNA formulations, including combinations of miRNA modulators, reveals complex patterns in macrophage-mediated immune responses under different conditions, such as hypoxia.
Abstract #54
Investigating the interplay between gut microbiome, monocytes/macrophages, and osteoarthritis
Atoosa Ziyaeyan, University of Toronto Faculty of Engineering
Background & Aim
Osteoarthritis (OA) is a complex degenerative joint disease influenced by various factors, including gut dysbiosis. This research aims to unravel the complex connection between metabolites produced by intestinal commensal bacteria and their impact on local immune effectors within the joint. The synovial joint is not a sterile environment, as shown by elevated levels of bacterial metabolites, such as lipopolysaccharide (LPS), correlating with OA severity. We hypothesize that gut metabolites influence local and systemic immune effectors, specifically monocytes/macrophages (MΦ), contributing to OA pathogenesis. To test this hypothesis, we are measuring the correlation of levels of short-chain fatty acids (SCFAs) which are bacterial metabolites, LPS, LPS-binding protein (LBP) with i) patient-reported outcomes (PROMs) and ii) with frequencies of classical (CD14+CD16low), intermediate (CD14+CD16+) and non-classical (CD14lowCD16+) MΦ subsets.
Methods, Results & Conclusion
LBP levels are measured by an ELISA assay, and a Kinetic-QCL Kinetic Chromogenic LAL Assay measures LPS levels. The level of SCFAs is measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The MΦ populations in the synovial fluid are investigated by flow cytometry. With an N=78, there was a significant negative correlation between the SF LBP and the frequency of intermediate and non-classical MΦ in SF, indicating that patients with higher levels of LBP in their SF had lower frequencies of intermediate and non-classical MΦ in their SF. In addition, there was a significant positive correlation between LBP levels in the plasma and intermediate MΦ, non-classical MΦ in SF, and the patient's body mass index (BMI). 34 out of 78 patients had BMIs ≥ 30 (considered obese). In these patients, we saw a positive correlation between LBP levels in SF and WOMAC-Function, showing that patients with higher LBP levels in their plasma were more functionally impaired. Of the 78 patients in our study, 43 were females, and 35 were males. There was a significant negative correlation between LBP levels in SF and SF intermediate MΦ frequency in males and females. In conclusion, the observed correlations in the patients suggest that gut metabolites, specifically LBP, have systemic effects on OA severity.